Despite awareness of brucellosis for more than two millennia, identification and treatment of human illness is documented only within the last century. Reduction in animal disease has been used strategically to reduce human disease. Treatment of human infection relies upon antibiotic therapies, but relapse is not uncommon and with the advent of bioengineering, the ability to introduce antibiotic resistance into Brucella may negate the only method for direct treatment of human brucellosis. Brucellosis in humans can affect a number of different tissues, but is most typically associated with the lympho-histiocytic disease that if left untreated invades other tissues and can kill the host. Disease depends upon the ability of the organism to survive intracellularly, and includes persistence within professional phagocytic cells. Protective immunity in the host requires both the humoral and cellular responses, and although much effort has been invested in the development of subunit vaccines there has been little of success along these lines. The safety of currently available vaccine strains for human use is questionable, since these are often used to prevent abortion with less concern for protection against infection. The aim of the work proposed is the development of improved vaccines based on attenuated intracellular survival to minimize persistence of the organism while stimulating a protective immune response. Signature-tagged mutagenesis will be used to identify mutants of attenuated virulence in the mouse model and the defect in intracellular survival will be verified in vitro in human macrophages. The mouse model of infection will be employed, since survival and persistence of the pathogen in this model relies upon intracellular survival in macrophages. Similarities in survival of the organism, disease and organ involvement support the use of the mouse model for the study of human brucellosis. Vaccination in mice will be evaluated for protection against both intraperitoneal and oral challenge. Many of the gene products identified may serve as targets for new therapeutic regimens, but that is beyond the scope of the current proposal.